Prior to diagnosis of type 2 diabetes (DM2) the state of insulin resistance (InsR) exists. While InsR is characterized by high plasma levels of insulin and glucose, it is hyperglycemia that is most often studied in the etiology of observed changes in microvascular dysfunction. Independent of its role in glucose metabolism insulin can alter the major endothelial cell (EC) function: exchange. How exchange changes also depends on sex. Specifically, acute high insulin (10-7 M, Ins+) increases microvascular permeability (Ps) to protein only in males. Further, EC from males and females retain sex differences in culture in the absence of oscillating reproductive hormones and acute (min) and chronic (weeks) exposure to insulin elicits both sex-dependent and -independent changes in EC signaling. These data lead to the formulation of the novel hypothesis, to be tested in 3 aims that insulin, in a genomic, sex-dependent manner, contributes to fluid balance via regulation of microvessel permeability (Ps) to proteins. Thus, Psalbumin in adult rats of both sexes will be assessed using quantitative in vivo and in vitro methods to determine the sex-dependence of acute insulin in the regulation of in situ microvascular exchange in Aim 1, when Ins+ is predicted to alter acutely (min-hrs) protein flux in males, not females. In Aim 2 whether sex differences in insulin action on Psalbumin are exacerbated during chronic hyperinsulinemia (Ins+, in the absence of confounding factors present in InsR and DM2) will be determined. Chronic insulin is predicted to lead to insidious changes in interstitial milieu manifested as changes in protease activity, elevated tissue protein and inflammatory mediators, and changes in EC phenotype with respect to levels of NO and endothelin-1 (ET-1). Finally, in Aim 3, to round out the vertical integration, cultured microvessel EC of males and females, respectively, will be used to determine which EC insulin receptor-mediated signaling mechanisms are sex-specific. The EC phenotype in response to insulin is posited to reflect M/F-differences in the contribution of the EC- insulin signaling pathways leading to endothelin (ET-1) and nitric oxide (NO) production, respectively. DM2 is a chronic progressive disease with a greater morbidity and mortality in women than men albeit the incidence is sex-independent. Knowledge of a) the direct role of Ins+ in loss of barrier function, b) the sex-specific, genomic EC responses to acute and chronic insulin, and c) the cellular Ins signaling mediating these responses will facilitate formulation of strategies for earler disease identification and tailoring of patient therapy to ameliorate, if not reverse, or stop disease progression.